Several stem cell-based reproductive technologies are in the research pipeline that may be used to restore the fertility of male patients who are rendered infertile by radiation or chemotherapy treatments for cancer or other non-malignant conditions. The promise of these technologies has prompted several academic research centers, including our Fertility Preservation Program in Pittsburgh to begin freezing testicular tissue (with IRB approval) for patients prior to initiating gonadotoxic treatments. The primary target patient population is prepubertal boys who are not producing sperm and currently have no options to preserve their future fertility. Pre-clinical studies are urgently needed to demonstrate the feasibility and safety of experimental stem cell therapies and accelerate their translation to the fertility clinic. We have developed a nonhuman primate model that is infertile due to high dose chemotherapy. We will use that model in the current application to test the hypothesis that SSCs in prepubertal testicular tissue can generate spermatogenesis and/or haploid gametes following autologous SSC transplantation or testicular tissue grafting. Aim 1 will determine the optimal timing for autologous SSC transplantation (prepuberty, puberty or adult) after prepubertal chemotherapy exposure to inform patient decision making. Aim 2 will optimize freezing conditions and generate haploid germ cells from autologous testicular tissue grafts and test their function by fertilizing monkey oocytes and establishing pregnancies. Aim 3 will develop the state-of-the-art for maintaining and expanding SSC numbers in culture. SSC culture will provide a powerful tool for dissecting the mechanisms that regulate SSC function in higher primates that are not amenable to transgenic approaches. With SSC culture it may also be possible to amplify the small number of SSCs that can be obtained from the testicular biopsy of a prepubertal boy to large numbers sufficient to initiate robust spermatogenesis after transplantation. The nonhuman primate model is relevant to human testis anatomy and physiology and monkeys model the concept of prepuberty, which is not present in rodents. We anticipate that the proposed studies will generate essential pre-clinical data to justify the translation of spermatogonial stem cell-based therapies to the clinic. RELEVANCE (See instructions): Chemotherapy and radiation treatments can cause permanent infertility and there are currently no options to preserve the fertility of prepubertal boys who are not producing sperm. This is a significant human health concern because over 50,000 people under the age of 20 are diagnosed with cancer each year in the United States and with improved therapies, most will survive. This project will provide valuable pre-clinical evidence that spermatogonial stem cells can preserve and restore fertility after gonadotoxic therapies.